The Eph receptor tyrosine kinases have emerged as a new important family of cancer targets. Many Eph receptors are frequently upregulated in cancer cells and some of them are also present in the tumor microenvironment. Furthermore, modulating the activity of these receptors has been shown to affect tumor growth and metastatic spread. Several studies have demonstrated that disrupting the binding of Eph receptors to their ligands, the ephrins, in preclinical mouse tumor models results in decreased tumor growth, which is likely due at least in part to inhibition of tumor angiogenesis. However, Eph-based anticancer strategies will have to take into account the multiplicity of mechanisms through which Eph receptors affect the malignant properties of cancer cells. Furthermore, it will be important to determine the characteristics of tumors that are most sensitive to inhibitors of Eph-ephrin binding versus those of tumors where these inhibitors may not be as effective. Binding interactions between Eph receptors and their ligands, the ephrins, are highly promiscuous. Despite this promiscuity in the recognition of their natural ephrin ligands, our work in collaboration with other components of this program has revealed that the high affinity ephrin-binding pockets of different Eph receptors show selectivity for artificial ligands such as chemical compounds and peptides, demonstrating that exquisitely specific targeting of individual Eph receptors can be achieved. The proposed studies aim to evaluate the effects of inhibitors of Eph receptor-ephrin binding on endothelial cells and cancer cells, determine whether the effects on cancer cells depend on the cellular context, and identify the characteristics of the tumor cells for which these inhibitors are most effective. This information will allow matching of particular Eph-based therapeutic strategies to the tumor characteristics. Cultured cancer cell lines will be used to evaluate the activities of monomeric soluble forms of Eph receptor ligand binding domains as prototypes for inhibitors of Eph receptor-ephrin binding. Furthermore, we will evaluate in cell culture and tumor xenografts Eph receptor-targeting small molecules and peptides to be collaboratively developed with the other components of the program project In particular, structural information will be used to improve the binding aftlnlty of previously identified small molecule and peptide leads that target the ligand-binding pocket of Eph receptors and inhibit ephrin binding.